RECENT FINDINGS: STRUCTURAL AND FUNCTIONAL STUDIES OF ORF1p - ORF1p is one of two L1 encoded proteins. Earlier studies by others of mouse ORF1p showed that it binds nucleic acids, acts as a nucleic acid chaperone, and forms trimers via a highly conserved coiled coil domain. However, the function of ORF1p in retrotransposition is largely unknown. We are using several approaches to examine this problem including analysis of the structural, biochemical, and biological effects of adaptive evolution, which involved mainly the coiled coil domain (Boissinot, et al, Mol. Biol. Evol. 18: 2186). To do so we resuscitated a pre-adapted ORF1p from an extinct L1 family, L1Pa5, which is ancestral to the adapted human(h) ORF1p of the currently active human L1Pa1 family. We also created mosaic versions of ORF1p that contain modern and ancestral regions, and other variants that were deleted of various domains. We extensively characterized these proteins with respect to retrotransposition and interaction with several mammalian host protein in vivo. In addition, we purified mg amounts of some the various of ORF1ps to homogeneity. We examined their various biophysical and biochemical properties in vitro including several assays that reflect their nucleic acid chaperone activity. We published two important and novel findings on the modern ORF1p this year: In particular, we unexpectedly found that trimeric hORF1p molecules polymerize under the very conditions that are conducive to high affinity nucleic acid binding, and that we can control the extent of polymerization by altering solution conditions. We also found that while hORF1p protects mismatched double-stranded nucleic acids from dissociation (melting) at low concentrations, at high concentrations (when largely polymeric), it melts nucleic acids. A mismatched duplex is a proxy nucleic acid chaperone substrate. Thus, determining the biophysical basis the surprising and novel diphasic affect of the protein on this substrate is essential to our further progress in this area. To this end we have established a collaborative project with Dr. Mark Williams to study these interactions by atomic force microscopy. (percentage of PI's Intramural Research Budget for this project for this year)